T5 bacteriophage has been shown to contain genetic information for the synthesis of at least 24 different tRNA species, including isoacceptor tRNAs. A physical DNA map has been constructed which indicates that the identified tRNA genes are clustered in six regions in the C segment of T5 DNA. One of the major objectives of this work is to further refine the DNA map so that the sequential order of the tRNA genes in their respective cluster is elucidated. This additional refinement will be carried out by restriction endonuclease mapping. We have started a new project concerned with the regulation of tRNA synthesis in rat tissue. To effectively study this problem, we have been using two-dimensional gel electrophoresis to resolve 32P-tRNA species derived from rat liver. So far, over 60 radioactive spots have been detected following gel electrophoresis, and we are currently in the process of identifying the tRNA species contained in these spots. Following identification, the purity of each tRNA specie will be determined by RPC-5 and finger-technique to determine whether changes in tRNA species production in various rat tissues are observed under different physiological conditions (i.e., immature vs. mature animals) and after the administration of certain chemical agents (i.e., hormones, chemical carcinogens, virsuses, etc.).